Proteases from gram-positive organisms

ABSTRACT

The present invention relates to the identification of novel metallo-proteases (MP) in Gram-positive microorganisms. The present invention provides the nucleic acid and amino acid sequences for Bacillus (MP). The present invention also provides host cells having mutation or deletion of part or all of the gene encoding MP. The present invention also provides host cells further comprising nucleic acid encoding desired heterologous proteins such as enzymes. The present invention also provides cleaning compositions comprising an MP of the present invention.

RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application fromPCT/US98/18828, filed Sep. 8, 1998, which claims priority under 35U.S.C. 119 of Great Britain Application No. 9719636.4, filed Oct. 15,1997, the disclosure of both applications are fully incorporated hereinby reference.

FIELD OF THE INVENTION

The present invention relates to metallo-proteases derived fromgram-positive microorganisms. The present invention provides nucleicacid and amino acid sequences of a metallo-protease identified inBacillus subtilis. The present invention also provides methods for theproduction of the protease in host cells as well as the production ofheterologous proteins in a host cell having a mutation or deletion ofpart or all of the proteases of the present invention.

BACKGROUND OF THE INVENTION

Gram-positive microorganisms such as members of the group Bacillus, havebeen used for large-scale industrial fermentation due, in part, to theirability to secrete their fermentation products into the culture media.In gram-positive bacteria, secreted proteins are exported across a cellmembrane and a cell wall, and then are subsequently released into theexternal media usually maintaining their native conformation.

Various gram-positive microorganisms are known to secrete extracellularand/or intracellular protease at some stage in their life cycles. Manyproteases are produced in large quantities for industrial purposes. Anegative aspect of the presence of proteases in gram-positive organismsis their contribution to the overall degradation of secretedheterologous or foreign proteins.

The classification of proteases found in microorganisms is based ontheir catalytic mechanism which results in four groups: the serineproteases; metallo-proteases; cysteine proteases; and asparticproteases. These categories, in general, can be distinguished by theirsensitivity to various inhibitors. For example, the serine proteases areinhibited by phenylmethylsulfonylfluoride (PMSF) anddiisopropylfluorophosphate (DIFP); the metallo-proteases by chelatingagents; the cysteine enzymes by iodoacetamide and heavy metals and theaspartic proteases by pepstatin. The serine proteases have alkaline pHoptima, the metalloproteases are optimally active around neutrality, andthe cysteine and aspartic enzymes have acidic pH optima (BiotechnologyHandbooks. Bacilluis. vol. 2. edited by Harwood, 1989 Plenum Press, NewYork).

Metallo-proteases form the most diverse of the catalytic types ofproteases. Family M23 contains bacterial enzymes such as the β-lyticendopeptidases of Lysobacter and

Achromobacier and the Pseudomonas LasA protein and have specificity forGly bonds, especially in Gly-GIy+Xaa-sequences (Methods in Enzymology,vol. 248, Academic Press, Inc. 1994). The enzymes of the M23 familycontain zinc and a conserved His-Xaa-His motif

SUMMARY OF THE INVENTION

The present invention relates to the discovery of a heretofore unknownmetallo-protease (MP) found in gram positive microorganisms, uses of theMP in industrial applications, and advantageous strain improvementsbased on genetically engineering such microorganisms to delete,underexpress or overexpress that MP. Due to the overall relatedness ofMP with Psezidomonas as lasA protein, including the presence of themotif His-Xaa-His, MP appears to be a member of the metallo-proteasefamily M23.

Applicant's discovery, in addition to providing a new and usefulprotease and methods, of detecting DNA encoding such proteases in a grampositive microorganism, provides several advantages which may facilitateoptimization and/or modification of strains of gram positivemicroorganisms, such as Bacillus, for expression of desired, e.g.heterologous, proteins. Such optimizations, as described below indetail, allow the construction of strains having decreased proteolyticdegradation of desired expression products.

Applicant's invention is further based on the discovery of the presenceof MP's in Gram-positive microorganisms. The Gram-positive microorganismmay be Bacillus and may also be selected from the group consisting ofBacillus stblilis, Bacillis stearotherniophilus, Bacillus lichenformisawd Bacillus amyloliqufaciens. The present invention further relies orthe discovery that naturally occurring MP is encoded by nucleic acidfound about 2248 kb from the point of origin of Bacillus subtilis I-168strain (Bacillus Genetic Stock Center, accession number 1A1, Columbus,Ohio). The present invention relates to the MP encoded thereby, as wellas the nucleic acid and amino acid molecules having the sequencesdisclosed in FIGS. 1A-1O (nucleic acid shown in SEQ ID NO:1; amino acidsequence shown in SEQ ID NO2).

The present invention thus provides methods for detecting gram positivemicroorganism homologs of B. stiblilis MP that comprises hybridizingpart or all of the nucleic acid encoding B. suzblilis MP with nucleicacid derived from gram-positive organisms, either of genomic or cDNAorigin. Accordingly, the present invention provides a method fordetecting a gram-positive microorganism MP, comprising the steps ofhybridizing gram-positive microorganism nucleic acid under lowstringency conditions to a probe, wherein the probe comprises part orall of the nucleic acid sequence shown in FIGS. 1A-1O (SEQ ID NO:1); andisolating gram-positive nucleic acid which hybridizes to said probe.

In a preferred embodiment, the Bacillus is selected from the groupconsisting of B. licheniformis, B. lenius, B. brevis, B.stearothermophilus, B. alkalophilts, B. amyloliquefaciens, B.coaguilmis, B. circiIlans, B. lautus and B. thuringiensis.

The production of desired heterologous proteins or polypeptides ingram-positive microorganisms may be hindered by the presence of one ormore proteases, including MP, which degrade the produced heterologousprotein or polypeptide. One advantage of the present invention is thatit provides methods and expression systems which can be used to preventthat degradation, thereby enhancing yields of the desired heterologousprotein or polypeptide. Accordingly, the present invention provides agram-positive microorganism having a mutation or deletion of part or allof the gene encoding MP, which results in the inactivation of the MPproteolytic activity, either alone or in combination with mutations inother proteases, such as apr, npr, epr, mpr, bpf or isp for example, orother proteases known to those of skill in the art. In one embodiment ofthe present invention, the gram-positive organism is a member of thegenus Bacillhs. In another embodiment, the Bacillus is selected from thegroup consisting of B. sitbtilis, B. lichentformis, B. lentizs, B.brevis, B. stearolhermophilus, B. alkalophiltis, B. amyloliquefaciens,B. coagulais, B. circulats, B. lautus and Bacillus thuringiensis. In afurther preferred embodiment, the Bacillus is Bacillus subtilis.

In another aspect, the gram-positive host having one or moremetallo-protease deletions or mutations is further geneticallyengineered to produce a desired protein. In one embodiment of thepresent invention, the desired protein is heterologous to thegram-positive host cell. In another embodiment, the desired protein ishomologous to the host cell. The present invention encompasses agram-positive host cell having a deletion, mutation or interruption ofthe nucleic acid encoding the naturally occurring homologous protein,such as a protease, and having nucleic acid encoding the homologousprotein re-introduced in a recombinant form. In another embodiment, thehost cell produces the homologous protein. Accordingly, the presentinvention also provides methods and expression systems for reducingdegradation of heterologous proteins produced in gram-positivemicroorganisms. The gram-positive microorganism may be normallysporulating or non-sporulating. In a preferred embodiment, the grampositive host cell is a Bacillus. In another preferred embodiment, theBacillus host cell is Bacillus. In another embodiment, the Bacillus isselected from the group consisting of B. subtilis, B. licheniformis, B.lennus, B. brevis, B. stearothermophilis, B. alkalophilus, B.amyloliquefaciens, B. coagulans, B. circulans, B. lautus and Bacillusthurgiensis.

Naturally occurring gram positive MP as well as proteolytically activeamino acid vanations or derivatives thereof, have application in thetextile industry, in cleaning compositions and in animal feed. Themetalloprotease MP may be used alone or in combination with otherenzymes and/or mediators or enhancers. Accordingly, in a further aspectof the present invention, gram-positive MP is produced on an industrialfermentation scale in a microbial host expression system. The. presentinvention provides a cleaning composition comprising a metalloprotease,MP, having the amino acid sequence shown in FIGS. 1A-1O (SEQ ID NO:2) orthe amino acid encoded by the MP nucleic acid found at about 2248kilobases from the point of origin of Bacillis subtilis. Also providedare cleaning compositions comprising a metalloprotease having at least80%, at least 90%, or at least 95% homology with the amino acid sequenceshown in FIGS. 1A-1O (SEQ ID NO:2) or comprising a metalloproteaseencoded by a gene that hybridizes with the nucleic acid shown in FIGS.1A-1O (SEQ ID NO:1) under high stringency conditions.

Further there is provided an animal feed comprising a metalloprotease,MP, having the amino acid sequence shown in FIGS. 1A-1O (SEQ ID NO:2).Also provided are animal feeds comprising a metalloprotease having atleast 80%, at least 90%, and at least 95% homology with the amino acidsequence shown in FIGS. 1A-1O (SEQ ID NO:2) or comprising ametalloprotease encoded by a gene that hybridizes with the nucleic acidshown in FIGS. 1A-1O (SEQ ID NO:1) under high stringency conditions.

Also provided is a composition for the treatment of a textile comprisinga metalloprotease, MP, having the amino acid sequence shown in FIGS.1A-1O (SEQ ID NO:2). Also provided are compositions for the treatment ofa textile comprising a metalloprotease having at least 80%, at least90%, or at least 95% homology with the amino acid sequence shown inFIGS. 1A-1O (SEQ ID NO:2) or comprising a metalloprotease encoded by agene that hybridizes with the nucleic acid shown in FIGS. 1A-1O (SEQ IDNO:2) under high stingency conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1O (SEQ ID NO:1) shows the DNA and amino acid sequence (SEQ IDNO:2) for Bacillus subtilis MP.

FIG. 2 show an amino acid alignment of Bacillus subtilis MP (designatedas YOMI) and Psetidomonas LasA. The amino acid motif H-X-H is noted atamino acid 308-310 in LasA.

DETAILED DESCRIPTON OF THE PREFERRED EMBODIMENTS Definitions

As used herein, the genus Bacillus includes all members known to thoseof skill in the art, including but not limited to B. subtilis, B.licheniformis, B. lenlus, B. brevis, B. stearolhermophilus, B.alkalophilus, B. amvliohquefociens, B. coguagtias, B. czculam, B. lautusand B thuringiensis.

The present invention relates to a newly characterized metallo-protease(MP) from gram positive organisms. In a preferred embodiment, themetallo-protease is obtainable from a gram-positive organism which is aBacilliis. In another preferred emibodiment, the metallo-protease isobtainable from a Bacillus which is selected from the group consistingof B. subtilis, B. lichernforms. B. lentus, B. brevis, B.stearothermophilus, B. alkalophiluis, B. amyloliquiefaciens. B.coaguldans, B. ciculans, B. laututs and B. thuringiensis.

In another preferred embodiment, the gram-positive organism is Bacillussubtilis and MP has the amino acid sequence encoded by the nucleic acidmolecule having the sequence that occurs around 2248 kilobases from thepoint of origin of Bacillus subtilis I-168.

In another preferred embodiment, Bacillus subtilis has the nucleic acidand amino acid sequence as shown in FIGS. 1A-1O (nucleic acid shown inSEQ ID NO:1; amino acid sequence shown in SEQ ID NO:2). The presentinvention encompasses the use of amino acid variations of the amino acidsequences disclosed in FIGS. 1A-1O (SEQ ID NO:2) that have proteolyticactivity. Such proteolytic amino acid variants can be used in thetextile industry, animal feed and in cleaning compositions. The presentinvention also encompasses the use of B. subtilis amino acid variationsor derivatives that are not proteolytically active. DNA encoding suchvariants can be used in methods designed to delete or mutate thenaturally occurring host cell MP.

As used herein “nucleic acid” refers to a nucleotide or polynucleotidesequence, and fragments or portions thereof, and to DNA or RNA ofgenomic or synthetic origin which may be double-stranded orsingle-stranded, whether representing the sense or antisense strand. Asused herein “amino acid” refers to peptide or protein sequences orportions thereof. A “polynucleotide homolog” as used herein refers to agram-positive microorganism polynucleotide that has at least 80%, atleast 90% and at least 95% identity to B. subtilis MP, or which iscapable of hybridizng to B. subtilis MP under conditions of highstringency and which encodes an amino acid sequence havingmetallo-protease activity.

The terms “isolated” or “purified” as used herein refer to a nucleicacid or amino acid that is removed from at least one component withwhich it is naturally associated.

As used herein, the term “heterologous protein” refers to a protein orpolypeptide that does not naturally occur in a gram-positive host cell.Examples of heterologous proteins include enzymes such as hydrolasesincluding proteases, cellulases, amylases, carbohydrases, and lipases;isomerases such as racemases, epimerases, tautomerases, or mutases;transferases, kinases and phophatases. The heterologous gene may encodetherapeutically significant proteins or peptides, such as growthfactors, cytokines, ligands, receptors and inhibitors, as well asvaccines and antibodies. The gene may encode commercially importantindustrial proteins or peptides, such as proteases, carbohydrases suchas amylases and glucoamylases, cellulases, oxidases and lipases. Thegene of interest may be a naturally occurring gene, a mutated gene or asynthetic gene.

The termr “homologous protein” refers to a protein or polypeptide nativeor naturally occurring in a gram-positive host cell. The inventionincludes host cells producing the homologous protein via recombinant DNAtechnology. The present invention encompasses a gram-positive host cellhaving a deletion or interruption of the nucleic acid encoding thenaturally occurring homologous protein, such as a protease, and havingnucleic acid encoding the homologous protein re-introduced in arecombinant form. In another embodiment, the host cell produces thehomologous protein.

As used herein, the term “overexpressing” when referring to theproduction of a protein in a host cell means that the protein isproduced in greater amounts than its production in its naturallyoccurring environment.

As used herein, the phrase “proteolytic activity” refers to a proteinthat is able to hydrolyze a peptide bond. Enzymes having proteolyticactivity are described in Enzyme Nomenclature, 1992, edited WebbAcademic Press, Inc.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The unexpected discovery of the metallo-protease M23 family member,designated herein as MP, found in translated, uncharacterized B.subtilis genomic sequences provides a basis for producing host cells,expression methods and systems which can be used to prevent thedegradation of recombinantly produced heterologous proteins.

Accordingly, in a preferred embodiment, the host cell is a gram-positivehost cell that has a deletion or mutation in the naturally occurringnucleic acid encoding MP said mutation resulting in deletion orinactivation of the production by the host cell of the MP proteolyticgene product. The host cell may additionally be genetically engineeredto produced a desired protein or polypeptide.

It may also be desired to genetically engineer host cells of any type toproduce a gram-positive MP. Such host cells are used in large scalefermentation to produce large quantities of the protease which may beisolated or purified and used in cleaning products, such as detergents,in textile treatments and as animal feed additives.

I. MP Sequences

The nucleic acid sequence and amino acid sequence for Bacillus subtilisMP are shown in FIGS. 1A-1O (nucleic acid shown in SEQ ID NO:1; aminoacid acid sequence shown in SEQ ID NO:2). As will be understood by theskilled artisan, due to the degeneracy of the genetic code, a variety ofpolynucleotides can encode the Bacillis subtilis MP having the aminoacid sequence shown in FIGS. 1A-1O (SEQ ID NO:2). The present inventionencompasses all such polynucleotides.

The present invention encompasses the use of MP polynucleotide homologsencoding gram-positive microorganism MPs which have at least 80%, or atleast 90% or at least 95% identity to B. subtilis MP shown in FIGS.1A-1O (SEQ ID NO:1) as long as the homolog encodes a protein that hasproteolytic activity.

Gram-positive polynucleotide homologs of B. subtilis MP may be obtainedby standard procedures known in the art from, for example, cloned DNA(e.g., a DNA “library”), genomic DNA libraries, by chemical synthesisonce identified, by cDNA cloning, or by the cloning of genomic DNA, orfragments thereof, purified from a desired cell. (See, for example,Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Glover,D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd.,Oxford, U.K. Vol. I, II.) A preferred source is from genomic DNA.

As will be understood by those of skill in the art, the polynucleotidesequence disclosed in FIGS. 1A-1O (SEQ ID NO:1) may reflect inadvertenterrors inherent to nucleic acid sequencing technology. Moreover, thesequence of polynucleotides derived from related species, e.g., otherBacillus, will contain variations to the sequences specificallydisclosed herein. Nonetheless, one of ordinary skill in the art is fullycapable of determining the correct sequences from the informationprovided herein regarding the invention. For example, as describedbelow, it is possible to identify the MP of the invention by virtue ofits location in the microorganism's genome. The present inventionencompasses the naturally occurring nucleic acid molecule having thenucleic acid sequence obtained from the genomic sequence of Bacillusspecies.

Nucleic acid encoding Bacillus subtilis MP starts around 2248 kilobasescounting from the point of origin in the Bacillus subtilis strain I-168(Anagnostopala, 1961, J. Bacteriol. 81: 741-746 or Bacillus GenomicStock Center, accession 1A1, Columbus, Ohio). The Bacillus subtilisplaint of origin has been described in Ogasawara, N. (1995, Microbiology141: Pt.2 257-59). Bacillus subtilis MP has a length of 2285 aminoacids. Based upon the location of the DNA encoding Bacillus subtilis MP,naturally occurring B. subtilis MP can be obtained by methods known tothose of skill in the art including PCR technology.

Oligonucleotide sequences or primers of about 10-30 nucleotides inlength can be designed from the polynucleotide sequence disclosed inFIGS. 1A-1O (SEQ ID NO:1) and used in PCR technology to isolate thenaturally occurring sequence from B. subtilis genomic sequences.

Another general strategy for the “cloning” of B. subtilis genomic DNApieces for sequencing uses inverse PCR. A known region is scanned for aset of appropriate restriction enzyme cleavage sites and inverse PCR isperformed with a set of DNA primers determined from the outermost DNAsequence. The DNA fragments from the inverse PCR are directly used astemplate in the sequencing reaction. The newly derived sequences can beused to design new oligonucleotides. These new oligonucleotides are usedto amplify DNA fragments with genomic DNA as template. The sequencedetermination on both strands of a DNA region is finished by applying aprimer walking strategy on the genomic PCR fragments. The benefit ofmultiple starting points in the primer walking results from the seriesof inverse PCR fragments with different sizes of new “cloned” DNApieces. From the most external DNA sequence a new round of inverse PCRis started. The whole inverse PCR strategy is based on the sequentialuse of conventional taq polymerase and the use of long range inverse PCRin those cases in which the taq polyrnerase failed to amplify DNAfragments. Nucleic acid sequencing is performed using standardtechnology. One method for nucleic acid sequencing involves the use of aPerkin-Elmer Applied Biosystems 373 DNA sequencer (Perkin-Elmer, FosterCity, Calif.).

Nucleic acid sequences derived from genomic DNA may contain regulatoryregions in addition to coding regions. Whatever the source, the isolatedMP gene should be molecularly cloned into a suitable vector forpropagation of the gene.

In the molecular cloning of the gene from genomic DNA, DNA fragments aregenerated, some of which will encode the desired gene. The DNA may becleaved at specific sites using various restriction enzymes.Alternatively, one may use DNAse in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, as for example,by sonication. The linear DNA fragments can then be separated accordingto size by standard techniques, including but not limited to, agaroseand polyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing the MP may be accomplished in a number of ways. Forexample, a B. subtilis MP gene of the present invention or its specificRNA, or a fragment thereof, such as a probe or primer, may be isolatedand labeled and then used in hybridization assays to detect agram-positive MP gene. (Benton, W. and Davis, R., 1977, Science 196:180;Grunstein, M. And Hogness, D., 1975, Proc. Natl. Acad. Sci. USA72:3961). Those DNA fragments sharing substantial sequence similarity tothe probe will hybridize under stringent conditions.

Accordingly, the present invention provides a method for the detectionof gram-positive MP polynucleotide homologs which comprises hybridizingpart or all of a nucleic acid sequence of B. subtilis MP withgram-positive microorganism nucleic acid of either genomic or cDNAorigin.

Also included within the scope of the present invention is the use ofgram-positive microorganism polynucleotide sequences that are capable ofhybridizing to the nucleotide sequence of B. subtilis MP underconditions of intermediate to maximal stringency. Hybridizationconditions are based on the melting temperature (Tm) of the nucleic acidbinding complex, as taught in Berger and Kimmel (1987, Guide toMolecular Cloninq Techniques, Methods in Enzymology, Vol 152, AcademicPress, San Diego Calif.) incorporated herein by reference, and confer adefined “strinyencv” as explained below.

“Maximum stringency” typically occurs at about Tm-5° C. (5° C. below theTm of the probe); “high stringency” at about 5° C. to 10° C. below Tm;“intermediate stringency” at about 10° C. to 20° C. below Tm; and “lowstringency” at about 20° C. to 25° C. below Tm. As will be understood bythose of skill in the art, a maximum stringency hybridization can beused to identif or detect identical polynucleotide sequences while anintermediate or low stringency hybridization can be used to identify ordetect polynucleotide sequence homologs.

The term “hybridization” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” (Coombs J (1994) Dictionary of Biotechnology, StocktonPress, New York N.Y.).

The process of amplification as carried out in polymerase chain reaction(PCR) technologies is described in Dieffenbach C W and G S Dveksler(1995, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press,Plainview N.Y.). A nucleic acid sequence of all least about 10nucleotides and as many as about 60 nucleotides from B. subtilis MPpreferably about 12 to 30 nucleotides, and more preferably about 20-25nucleotides can be used as a probe or PCR primer.

The B. subtilis MP amino acid sequences (shown in FIGS. 1A-1O, SEQ IDNO:2) were identified via a BLAST search (Altschul, Stephen, Basic localalignment search tool, J. Mol. Biol. 215:403-410) of Bacillus subtilisgenornic nucleic acid sequences. B. subtilis MP (YOMI) was identified byits overall nucleic acid identity to the metallo-protease, PseudomonaslasA, including the presence of the catalytic domain H-X-H as shown inFIG. 2.

II. Expression Systems

The present invention provides host cells, expression methods andsystems for the enhanced production and secretion of desiredheterologous or homologous proteins in gram-positive microorganisms. Inone embodiment, a host cell is genetically engineered to have a deletionor mutation in the gene encoding a gram-positive MP such that therespective activity is deleted In another embodiment of the presentinvention, a gram-positive microorganism is genetically engineered toproduce a metallo-protease of the present invention.

Inactivation of a gram-positive metallo-protease in a host cell

Producing an expression host cell incapable of producing the naturallyoccurring metallo-protease necessitates the replacement andlorinactivation of the naturally occurring gene from the genome of the hostcell. In a preferred embodiment, the mutation is a non-revertingmutation.

One method for mutating nucleic acid encoding a gram-positivemetallo-protease is to clone the nucleic acid or part thereof, modifythe nucleic acid by site directed mutagenesis and reintroduce themutated nucleic acid into the cell on a plasmid. By homologousrecombination, the mutated gene may be introduced into the chromosome.In the parent host cell, the result is that the naturally occurringnucleic acid and the mutated nucleic acid are located in tandem on thechromosome. After a second recombination, the modified sequence is leftin the chromosome having thereby effectively introduced the mutationinto the chromosomal gene for progeny of the parent host cell.

Another method for inactivating the metallo-protease proteolyticactivity is through deleting the chromosomal gene copy. In a preferredembodiment, the entire gene is deleted, the deletion occurring in suchas way as to make reversion impossible. In another preferred embodiment,a partial deletion is produced, provided that the nucleic acid sequenceleft in the chromosome is too short for homologous recombination with aplasmid encoded metallo-protease gene. In another preferred embodiment,nucleic acid encoding the catalytic amino acid residues are deleted.

Deletion of the naturally occurring gram-positive microorganismmetallo-protease can be carried out as follows. A metallo-protease geneincluding its 5′ and 3′ regions is isolated and inserted into a cloningvector. The coding region of the metallo-protease gene is deleted formthe vector in vitro, leaving behind a sufficient amount of the 5′ and 3′flanking sequences so to provide for homologous recombination with thenaturally occurring gene in the parent host cell. The vector is thentransformed into the gram-positive host cell The vector integrates intothe chromosome via homologous recombination in the flanking regions.This method leads to a gram-positive strain in which the protease genehas been deleted.

The vector used in an integration method is preferably a plasmid. Aselectable marker may be included to allow for ease of identification ofdesired recombinant microorgansims. Additionally, as will be appreciatedby one of skill in the art, the vector is preferably one which can beselectively integrated into the chromosome. This can be achieved byintroducing an if inducible origin of replication, for example, atemperature sensitive origin into the plasmid. By growing thetransformants at a temperature to which the origin of replication issensitive, the replication finction of the plasmid is inactivated,thereby providing a means for selection of chromosomal integrants.Integrants may be selected for growth at high temperatures in thepresence of the selectable marker, such as an antibiotic. Integrationmechanisms are described in WO 88/06623.

Integration by the Campbell-type mechanism can take place in the 5′flanking region of a the protease gene, resulting in a protease positivestrain carrying the entire plasmid vector in the chromosome in themetallo-protease locus. Since illegitimate recombination will givedifferent results it will be necessary to determine whether the completegene has been deleted, such as through nucleic acid sequencing orrestriction maps.

Another method of inactivating the naturally occurring metallo-proteasegene is to mutagenize the chromosomal gene copy by transforming agram-positive microorganism with oligonucleotides which are mutagenic.Alternatively, the chromosomal metallo-protease gene can be replacedwith a mutant gene by homologous recombination.

The present invention encompasses host cells having additional proteasedeletions or mutations, such as deletions or mutations in apr, npr, epr,mpr and others known to those of skill in the art.

One assay for the detection of mutants involves growing the Bacillushost cell on medium containing a protease substrate and measuring theappearance or lack thereof, of a zone of clearing or halo around thecolonies. Host cells which have an inactive protease will exhibit littleor no halo around the colonies.

III. Production of Metallo-protease

For production of metallo-protease in a host cell, an expression vectorcomprising at least one copy of nucleic acid encoding a gram-positivemicroorganism MP, and preferably comprising multiple copies, istransformed into the host cell under conditions suitable for expressionof the metallo-protease. In accordance with the present invention,polynucleotides which encode a gram-positive microorganism MP, orfragments thereof, or fusion proteins or polynucleotide homologsequences that encode amino acid variants of B. subtilis MP, may be usedto generate recombinant DNA molecules that direct their expression inhost cells. In a preferred embodiment, the gram-positive host cellbelongs to the genus Bacillus. In another preferred embodiment, the grampositive host cell is B. subtilis.

As will be understood by those of skill in the art, it may beadvantageous to produce polynucleotide sequences possessingnon-naturally occurring codons. Codons preferred by a particulargram-positive host cell (Murray E et al (1989) Nuc Acids Res 17:477-508)can be selected, for example, to increase the rate of expression or toproduce recombinant RNA transcripts having desirable properties, such asa longer half-life, than transcripts produced from naturally occurringsequence.

Altered MP polynucleotide sequences which may be used in accordance withthe invention include deletions, insertions or substitutions ofdifferent nucleotide residues resulting in a polynucleotide that encodesthe same or a functionally equivalent MP homolog, respectively. As usedherein a “deletion” is defined as a change in either nucleotide or aminoacid sequence in which one or more nucleotides or amino acid residues,respectively, are absent.

As used herein an “insertion” or “addition” is that change in anucleotide or amino acid sequence which has resulted in the addition ofone or more nucleotides or amino acid residues, respeetively, ascompared to the naturally occurring MP.

As used herein “substitution” results from the replacement of one ormore nucleotides or amino acids by different nucleotides or amino acids,respectively.

The encoded protein may also show deletions, insertions or substitutionsof amino acid residues which produce a silent change and result in afunctionally equivalent MP variant. Deliberate amino acid substitutionsmay be made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues as long as the variant retains the ability to modulatesecretion. For example, negatively charged amino acids include asparticacid and glutamic acid; positively charged amino acids include lysineand arginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine, valine;glycine, alanine; asparagine, glutamine; serine, threonine,phenylalanine, and tyrosine.

The MP polynucleotides of the present invention may be engineered inorder to modify the cloning, processing and/or expression of the geneproduct. For example, mutations may be introduced using techniques whichare well known in the art, eg, site-directed mutagenesis to insert newrestriction sites, to alter glycosylation patterns or to change codonpreference, for example.

In one embodiment of the present invention, a gram-positivemicroorganism MP polynucleotide may be ligated to a heterologoussequence to encode a fusion protein. A fusion protein may also beengineered to contain a cleavage site located between themetallo-protease nucleotide sequence and the heterologous proteinsequence, so that the metallo-protease may be cleaved and purified awayfrom the heterologous moiety.

IV. Vector Sequences

Expression vectors used in expressing the metallo-proteases of thepresent invention in gram-positive microorganisms comprise at least onepromoter associated with a metallo-protease selected from the groupconsisting of MP, which promoter is functional in the host cell. In oneembodiment of the present invention, the promoter is the wild-typepromoter for the selected metallo-protease and in another embodiment ofthe present invention, the promoter is heterologous to themetallo-protease, but still functional in the host cell. In onepreferred embodiment of the present invention, nucleic acid encoding themetallo-protease is stably integrated into the microorganism genome.

In a preferred embodiment, the expression vector contains a multiplecloning site cassette which preferably comprises at least onerestriction endonuclease site unique to the vector, to facilitate easeof nucleic acid manipulation. In a preferred embodiment, the vector alsocomprises one or more selectable markers. As used herein, the termselectable marker refers to a gene capable of expression in thegram-positive host which allows for ease of selection of those hostscontaining the vector. Examples of such selectable markers include butare not limited to antibiotics, such as, erythromycin, actinomycin,chloramphenicol and tetracycline.

V. Transformation

A variety of host cells can be used for the production Bacillus subtilisMP or MP homologs including bacterial, fungal, mammalian and insectscells. General transformation procedures are taught in Current ProtocolsIn Molecular Biology (vol. 1, edited by Ausubel et al., John Wiley &Sons, Inc. 1987, Chapter 9) and include calcium phosphate methods,transformation using DEAE-Dextran and electroporation. Planttransformation methods are taught in Rodriquez (WO 95/14099, publishedMay 26, 1995).

In a preferred embodiment, the host cell is a gram-positivemicroorganism and in another preferred embodiment, the host cell isBacillis. In one embodiment of the present invention, nucleic acidencoding one or more MP(s) of the present invention is introduced into ahost cell via an expression vector capable of replicating within theBacillus host cell. Suitable replicating plasmids for Bacillus aredescribed in Molecular Biological Methods for Bacillus, Ed. Harwood andCutting, John Wiley & Sons, 1990, hereby expressly incorporated byreference; see chapter 3 on plasmids. Suitable replicating plasmids forB. subtilis are listed on page 92.

In another embodiment, where it is desired to produce the MP for use incleaning compositions, nucleic acid encoding MP is stably integratedinto the microorganism genome. Preferred host cells are gram-positivehost cells. Another preferred host is Bacillus. Another preferred hostis Bacillus subtilis. Several strategies have been described in theliterature for the direct cloning of DNA in Bacillus. Plasmid markerrescue transformation involves the uptake of a donor plasmid bycompetent cells carrying a partially homologous resident plasmid(Contente et al., Plasmid 2:555-571 (1979); Haima et al., Mol. Gen.Genet. 223:185-191 (1990); Weinrauch et al., J. Bacteriol.154(3):1077-1087 (1983); and Weinrauch et al., J. Bacteriol.169(3):1205-1211 (1987)). The incoming donor plasmid recombines with thehomologous region of the resident “helper” plasmid in a process thatmimics chromosomal transformation.

Protoplast transformation is described for B. subtilis in Chang andCohen, (1979) Mol. Gen. Genet 168:111-115; for B. megaterium inVorobjeva et al., (1980) FEMS Microbiol. Letters 7:261-263; for B.amylolquefaciens in Smith et al., (1986) AppI. and Env. Microbiol.51:634; for B. thuringiensis in Fisher et al., (1981) Arch. Microbiol.139:213-217; for B. sphaericus in McDonald (1984) J. Gen. Microbiol.130:203; and B. larvae in Bakiet et al., (1985, Appi. Environ.Microbiol. 49:577). Mann et al., (1986, Current Microbiol. 13:131-135)report on transformation of Baciliis protoplasts and Holubova, (1985)Folia Microbiol. 30:97) disclose methods for introducing DNA intoprotoplasts using DNA containing liposomes.

VI. Identification of Transformants

Whether a host cell has been transformed with a mutated or a naturallyoccurring gene encoding a gram-positive MP, detection of thepresencelabsence of marker gene expression can suggest whether the geneof interest is present However, its expression should be confirmed. Forexample, if the nucleic acid encoding an MP of the present invention isinserted within a marker gene sequence, recombinant cells containing theinsert can be identified by the absence of marker gene function.Alternatively, a marker gene can be placed in tandem with nucleic acidencoding the MP under the control of a single promoter. Expression ofthe marker gene in response to induction or selection usually indicatesexpression of the MP as well.

Alternatively, host cells which contain the coding sequence for ametallo-protease and express the protein may be identified by a varietyof procedures known to those of skill in the art. These proceduresinclude, but are not limited to, DNA-DNA or DNA-RNA hybridization andprotein bioassay or immunoassay techniques which include membrane-based,solution-based, or chip-based technologies for the detection and/orquantification of the nucleic acid or protein.

The presence of the metallo-protease polynucleotide sequence can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes, portions or fragments of B. subtilis MP.

VII. Assay of Protease Activity

There are various assays known to those of skill in the art fordetecting and measuring protease activity. There are assays based uponthe release of acid-soluble peptides from casein or hemoglobin measuredas absorbance at 280 nm or calorimetrically using the Folin method(Bergmeyer, et al., 1984, Methods of Enzymatic Analysis vol. 5,Peptidases, Proteinases and their Inhibitors, Verlag Chemie, Weinheim).Other assays involve the solubilization of chromogenic substrates (Ward,1983, Proteinases, in Microbial Enzymes and Biotechnology (W. M.Fogarty, ed.), Applied Science, London, pp. 251-317).

VIII. Secretion of Recombinant Proteins

Means for determining the levels of secretion of a heterologous orhomologous protein in a gram-positive host cell and detecting secretedproteins include, using either polyclonal or monoclonal antibodiesspecific for the protein. Examples include enzyme-linked immunosorbentassay (ELISA), radioimmunoassay (RMA) and fluorescent activated cellsorting (FACS). These and other assays are described, among otherplaces, in Hampton R et al (1990, Serological Methods, a LaboratoryManual, APS Press, St Paul Minn.) and Maddox DE et al (1983, J Exp Med158:1211).

A wide variety of labels and conjugation techniques are known by thoseskilled in the is art and can be used in various nucleic and amino acidassays. Means for producing labeled hybridization or PCR probes fordetecting specific polynucleotide sequences include oligolabeling, nicktranslation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the nucleotide sequence, or any portion ofit, may be cloned into a vector for the production of an mRNA probe.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by addition of an appropriateRNA polymerase such as T7, T3 or SP6 and labeled nucleotides.

A number of companies such as Pharmacia Biotech (Piscataway N.J.),Promega (Madison Wis.), and US Biochemical Corp (Cleveland Ohio) supplycommercial kits and protocols for these procedures. Suitable reportermolecules or labels include those radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles and the like. Patents teachingthe use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. Also,recombinant immunoglobulins may be produced as shown in U.S. Pat. No.4,816,567 and incorporated herein by reference.

IX. Purification of Proteins

Gram positive host cells transformed with polynucleotide sequencesencoding heterotogous or homologous protein may be cultured underconditions suitable for the expression and recovery of the encodedprotein from cell culture. The protein produced by a recombinantgram-positive host cell comprising a mutation or deletion of themetallo-protease activity will be secreted into the culture media. Otherrecombinant constructions may join the heterologous or homologouspolynucleotide sequences to nucleotide sequence encoding a polypeptidedomain which will facilitate purification of soluble proteins (Kroll D Jet al (1993) DNA Cell Biol 12:441-53).

Such purification facilitating domains include, but are not limited to,metal chelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals (Porath J (1992) Protein Expr Purif3:263-281), protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle Wash.). The inclusion of acleavable linker sequence such as Factor XA or enterokinase (Invitrogen,San Diego Calif.) between the purification domain and the heterologousprotein can be used to facilitate purification.

X. Uses of The Present Invention

MP and Genetically Engineered Host Cells

The present invention provides genetically engineered host cellscomprising mutations, preferably non-revertable mutations, or deletionsin the naturally occurring gene encoding MP such that the proteolyticactivity is diminished or deleted altogether. The host cell may containA=additional protease deletions, such as deletions of the maturesubtilisn protease and/or mature neutral protease disclosed in U.S. Pat.No. 5,264,366.

In a preferred embodiment, the host cell is further geneticallyengineered to produce a desired protein or polypeptide. In a preferredembodiment the host cell is a Bacillis. In another preferred embodiment,the host cell is a Bacillus subtilis.

In an alternative embodiment, a host cell is genetically engineered toproduce a gram-positive MP. In a preferred embodiment, the host cell isgrown under large scale fermentation conditions. In another preferredembodiment, the MP is isolated and/or purified and used in the textileindustry, the feed industry and in cleaning compositions such asdetergents.

As noted, MP can be useful in formulating various cleaning compositions.A number of known compounds are suitable surfactants useful incompositions comprising the MP of the invention. These include nonionic,anionic, cationic, anionic or zwitterionic detergents, as disclosed inU.S. Pat. No. 4,404,128 and U.S. Pat. No. 4,261,868. A suitabledetergent formulation is that described in Example 7 of U.S. Pat. No.5,204,015. The art is familiar with the different formulations which canbe used as cleaning compositions. In addition, MP can be used, forexample, in bar or liquid soap applications, dishcare formulations,contact lens cleaning solutions or products, peptide hydrolysis, wastetreatment, textile applications, as fusion-cleavage enzymes in proteinproduction, etc. MP may comprise enhanced performance in a gdetergentcomposition (as compared to another detergent protease). As used herein,enhanced performance in a detergent is defined as increasing cleaning ofcertain enzyme sensitive stains such as grass or blood, as determined byusual evaluation after a standard wash cycle.

MP can be formulated into known powdered and liquid detergents having pHbetween 6.5 and 12.0 at levels of about 0.01 to about 5% (preferably0.1% to 0.5%) by weight. These detergent cleaning compositions can alsoinclude other enzymes such as known proteases, amylases, cellulases,lipases or endoglycosidases, as well as builders and stabilizers.

The addition of MP to conventional cleaning compositions does not createany special use limitation. In other words, any temperature and pHsuitable for the detergent is also suitable for the pr esentcompositions as lon as the pH is within the above range, and thetemperature is below the described MP's denaturing temperature. Inaddition, MP can be used in a cleaning composition without detergents,again either alone or in combination with builders and stabilizers.

Proteases can be included in animal feed such as part of animal feedadditives as described in, for example, U.S. Pat. No. 5,612,055; U.S.Pat. No. 5,314,692; and U.S. 5,147,642.

One aspect of the invention is a composition for the treatment of atextile that includes MP. The composition can be used to treat forexample silk or wool as described in publications such as RD 216,034; EP134,267, U.S. Pat. No. 4,533,359; and EP 344,259.

MP Polynucleotides

A B. subtlis MN polynucleotide, or any part thereof, provides the basisfor detecting the presence of gram-positive microorganism MPpolynucleotide homologs through hybridization techniques and PCRtechnology.

Accordingly, one aspect of the present invention is to provide fornucleic acid hybridization and PCR probes which can be used to detectpolynucleotide sequences, including genoaic and cDNA sequences, encodinggram-positive MP or por tions thereof. In another aspect ofthe presentinvention, an fe polynucleotide can be used in hybridization technologyto detect the major protease of a gram-positive microorganism due to theproximity of the MP with the major protease.

The manner and method of carrying out the present invention may be morefully understood by those of skill in the art by reference to thefollowing examples, which examples are not intended in any manner tolimit the scope of the present invention or of the claims directedthereto.

EXAMPLE I Preparation of a Genomic library

The following example illustrates the preparation of a Bacillus genomiclibrary.

Genomic DNA from Bacillus cells is prepared as taught in CurrentProtocols In so Molecular Biology vol. 1, edited by Ausubel et al., JohnWiley & Sons, Inc. 1987, chapter 2. 4.1. Generally, Bacillus cells froma saturated liquid culture are lysed and the proteins removed bydigestion with proteinase K. Cell wall debris, polysaccharides, andremaining proteins are removed by selective precipitation with CTAB, andhigh molecular weight genornic DNA is recovered from the resultingsupernatant by isopropanolprecipitation. If exceptionally clean genomicDNA is desired, an additional step of purifying the Bacillus genomic DNAon a cesium chloride gradient is added.

After obtaining purified genomic DNA, the DNA is subjected to Sau3Adigestion. Sau3A recognizes the 4 base pair site GATC and generatesfragments compatible with several convenient phage lambda and cosmidvectors. The DNA is subjected to partial digestion to increase thechance of obtaining random fragments.

The partially digested Bacillus genomic DNA is subjected to sizefractionation on a 1% agarose gel prior to cloning into a vector.Alternatively, size fractionation on a sucrose gradient can be used. Thegenomic DNA obtained from the size fractionation step is purified awayfrom the agarose and ligated into a cloning vector appropriate for usein a host cell and transformed into the host cell.

EXAMPLE II Detection of gram-positive microorganisms

The following example describes the detection of gram-positivemicroorganism MP.

DNA derived from a gram-positive microorganism is prepared according tothe methods disclosed in Current Protocols in Molecular Biology, Chap. 2or 3. The nucleic acid is subjected to hybridization and/or PCRamplification with a probe or primer derived from MP.

The nucleic acid probe is labeled by combining 50 pmol of the nucleicacid and 250 mCi of [gamma ³²P] adenosine triphosphate (Amersham,Chicago Ill.) and T4 polynucleotide kinase (DuPont NEN®, Boston Mass.).The labeled probe is purified with Sephadex G-25 super fine resin column(Pharmacia). A portion containing 10⁷ counts per minute of each is usedin a typical membrane based hybridization analysis of nucleic acidsample of either genomic or cDNA origin.

The DNA sample which has been subjected to restriction endonucleasedigestion is fractionated on a 0.7 percent agarose gel and transferredto nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH).Hybridization is carried out for 16 hours at 40 degrees C. To removenonspecific signals, blots are sequentially washed at room temperatureunder increasingly stringent conditions up to 0.1× saline sodium citrateand 0.5% sodium dodecyl sulfate. The blots are exposed to film forseveral hours, the film developed and hybridization patterns arecompared visually to detect polynucleotide homologs of B. subtilis MP.The homologs are subjected to confirmatory nucleic acid sequencing.Methods for nucleic acid is sequencing are well known in the art.Conventional enzymatic methods employ DNA polymerase Kienow fragment,SEQUENASE® (US Biochemical Corp, Cleveland, Ohio) or Taq polymerase toextend DNA chains from an oligonucleotide primer annealed to the DNAtemplate of interest.

Various other examples and modifications of the foregoing descriptionand examples will be apparent to a person skilled in the art afterreading the disclosure without departing from the spirit and scope ofthe invention, and it is intended that all such examples ormodifications be included within the scope of the appended claims. Allpublications and patents referenced herein are hereby incorporated byreference in their entirety.

3 1 7100 DNA Bacillius subtilis 1 atattggcat ggtgttatgg atgtaattattaagaaagca aacaaagtcg ctcaataact 60 gagtggcttt tttctttgtc ctctcccctactgaaaggaa gtgattctta cttgagtcaa 120 aacctcaaaa ttatactaac cccgcaagctgatacctcat ccaaaactgt cgaacagtta 180 aatcagcaaa ttaaatccct ggaaaagaaactcaactccc tcaagctcaa tacaaacatt 240 gattctacaa ccttaaaagc tctgcaagaattctcctctg ctatcgacac atatcagaaa 300 aacctaaaat cctataatca aacagttaaagaaacctcaa cagtaattaa gaatgctgac 360 ggatcagttg aaaagctcac ccagcagtataagaaaaatg gtgagatact tcaacgtgaa 420 acaaaaataa tcaacaatcg taatacagcattaaagcaag aaactcaaga ggttaacaag 480 ctaacacagg ccactgagaa actaggacaggttcaaaaaa agactgtgca gagaaatctg 540 caaggacagc caacaaaggt agtgcagaaaaaccgccacg ggttcgatga tattgtttat 600 acaactgatc ctaaaactaa ttcgacctcctcaaaaacta caactaatta tgaccaacaa 660 aggagagcaa ttgagcagct taagcaagatttagagaagc ttagacagca aggtattgtt 720 actgatacga ccatctcatc tcttggccgaaaaataaaca cagctcaatc cgctcaacaa 780 attgaagcac tgcaaaatag gataaggatgttagatgata aatctgcggc agttgcgaag 840 aacaatgaat taaagaaaac cattgaattatatcagcgac aggcacaagt aaatgttcaa 900 aacctaaata cacggtatgg cagttctatgggctctagta atagacaagc tgttcaagat 960 tatttgaatg cagtaaatag tcttaatgtaagcactggaa gcaataatat cagatcacaa 1020 attcaaagct tgaatatgca atttagagaattagcctcca acgctcaaac agctgctaat 1080 caagcctctt cttttggagc agaactaacccaaaccttca aaagcatgtc cacctattta 1140 atctccggtt ctttattcta cggagctatctctggactta aagaaatggt atcccaggca 1200 atagaaattg atactctcat gacaaatattcgccgtgtta tgaatgagcc ggattataaa 1260 tataatgaac ttctccaaga atctattgacttaggtgata cactttcaaa taaaatcaca 1320 gatattcttc aaatgacagg cgattttgggagaatgggtt tcgatgaaag tgagctctcc 1380 acgttaacga aaactgccca agttcttcaaaatgtctctg atttaactcc cgatgataca 1440 gttaacactc taacggcagc aatgctcaactttaatattg cagcaaatga ttcaatatca 1500 attgcagata aattaaatga ggttgataataactatgctg ttacaactct agatctggcc 1560 aattctatcc gtaaagctgg ttcaactgcttctacattcg gggtagagct aaatgatctt 1620 attggttata caactgcaat tgctagtacaacacgtgaat cagggaatat cgtcgggaac 1680 tccttaaaga caattttcgc gcggattgggaataatcaaa gctcaattaa agcgttagaa 1740 cagattggta tctcagttaa aacagctggtggtgaagcta aatcagcaag tgatttaatt 1800 agtgaagttg ctggtaagtg ggatacgctttctgatgctc agaaacaaaa tacttcaatt 1860 ggagtagctg gtatttatca attatcccgttttaatgcaa tgatgaacaa cttctctatt 1920 gctcagaatg cggctaaaac tgcggctaactcaacaggaa gtgcttggag tgagcagcaa 1980 aagtatgcag atagtctaca agctagggtaaataagcttc aaaataactt cactgaattt 2040 gctattgcag cttctgatgc ttttattagcgacggattaa ttgaatttac tcaagccgca 2100 ggttctttgc ttaacgcttc tacaggagtaatcaaatcag ttgggttcct acctcccctt 2160 ttagctgcag taagcactgc aacccttttgctcagtaaga atacccgcac attagccagc 2220 agcctaattt tgggcacacg tgcaatggggcaagaaactt tagcgactgc tgggctagaa 2280 gctggtatga ctcgtgcagc agtcgcctcaagagttctaa aaactgctct tcgagggttg 2340 cttgtttcaa ctttagttgg cggtgcatttgctgctttgg gatgggcgct agaatcatta 2400 atttcttctt ttgcagaagc taaaaaagctaaagatgatt ttgagcagag ccagcaaacc 2460 aatgtcgaag caattacgac caataaagactccactgata aactaataca gcaatataaa 2520 gagcttcaaa aagttaaaga gtcaagatctttaacttcag atgaagagca agaatacctt 2580 caagtcactc agcaattagc acaaactttccctgcattag ttaaaggcta tgattctcaa 2640 ggaaatgcaa ttcttaagac aaataaagagcttgaaaaag cgattgagaa tactaaagag 2700 tatttggctt taaagaaaca agaaacaagagacagcgcaa agaaaacatt cgaagacgct 2760 tctaaggaaa ttaaaaagtc taaggatgaattaaagcagt acaaacaaat agctgactac 2820 aacgataaag gtagacctaa atgggatctcattgcagatg acgatgacta taaggttgca 2880 gctgataaag ctaaacaaag tatgctcaaagctcaatctg acattgagag tggaaatgct 2940 aaagttaaag atagcgtcct ttcaattgcaaatgcttata gttcaattga tatcagtaat 3000 actttaaaga cgagtattag tgatgttgtcaacaaactta acttaaaaga tgatttagat 3060 cctgaagaat tagaaaaatt ctcctcttctttaggaaagc ttcaagaaaa aatgcaaaaa 3120 gctttagatt caggcgatga aaaagctttcgataacgcaa aaaaagatct tcaaagtctc 3180 ttggaaacat actccaaatc cgattcttctattgatgttt ttaaaatgag cttcgacaaa 3240 gcacagaaga acataaaaga tggagataagagcttatctt ccgtcaaatc tgaagttggt 3300 gatttaggtg agacgctggc agaagcaggtaacgaggcag aagattttgg taagaagcta 3360 aaagaagctc tggatgcaaa tagtgttgatgatattaagg cagctattaa agaaatgtca 3420 gatgctatgc agttcgattc cgttcaagatgtcttaaatg gggatatttt taataacacc 3480 aaagatcaag tagctcctct caatgatcttctggaaaaaa tggctgaagg taaaagtatt 3540 tctgcaaatg aagctaatac ccttattcaaaaagataagg aacttgccca ggctattagc 3600 atcgaaaatg gcgttgtgaa aattaaccgtgatgaagtta tcaaacaaag aaaagttaaa 3660 cttgatgctt ataacgacat ggttacctacagcaataaat tgatgaaaac agaagttaac 3720 aacgctatca aaactttaaa cgctgataccttacggattg acagcctgaa aaagctacga 3780 aaagaacgaa agcttgatat gtctgaggccgaactgtcag acctagaagt taagtcaatt 3840 aataatgttg cagatgcaaa aaaagaacttaaaaagcttg aagagaaaat gcttcaacct 3900 ggtggatact ccaatagtca aattgaagcaatgcaaagcg ttaaatcagc tttagaatct 3960 tatatttctg catctgaaga agccaccagtacacaagaaa tgaataaaca ggcacttgtt 4020 gaagctggaa catcattgga gaattggacagatcaacaag aaaaagccaa tgaagaaacc 4080 aagacttcca tgtatgttgt tgataaatacaaggaagcat tagaaaaagt taatgctgag 4140 attgacaagt acaacaagca ggtcaatgattatcctaaat actctcagaa atatcgagat 4200 gcaatcaaga aagaaattaa agcacttcagcaaaagaaaa agcttatgca ggaacaagct 4260 aagctgctta aagatcaaat taaatccggtaacattactc aatacggtat tgtaacctct 4320 acaacttctt ctggtggaac cccctcctcaactggtggat catattcagg caagtattca 4380 agctacataa attcagcagc tagtaaatacaatgttgacc ctgcccttat tgcagctgta 4440 attcagcaag aatcagggtt taatgctaaagcacgatctg gtgtaggtgc catgggatta 4500 atgcaactga tgccagcaac agcaaaaagcttaggagtaa ataacgctta cgatccttat 4560 caaaatgtta tgggtggaac aaagtacctcgcccaacaac ttgaaaagtt tggcggtaat 4620 gttgaaaaag cattggctgc atataatgctgggcctggta acgtaattaa atatggtggt 4680 atccctcctt ttaaagaaac acagaattacgtcaagaaga tcatggccaa ctatagcaaa 4740 tcgctctcat ctgccacttc ttcaatcgccagctattata caaataatag cgcttttagg 4800 gtaagctcca aatatggaca acaggaatctggtctccgct cctccccaca caaaggaact 4860 gattttgctg caaaagcagg tacagcaattaaatctcttc aaagtggtaa agtccaaatt 4920 gctggctaca gtaaaactgc aggtaactgggttgttatta aacaggatga tggaacagtt 4980 gccaagtaca tgcacatgct taacactccttctgtaaaag caggtcaatc agttaaagcc 5040 ggtcaaacta ttggtaaagt tggtagtacagggaactcga ctgggaacca ccttcattta 5100 cagatcgaac aaaatggaaa aacaatcgatcctgaaaagt acatgcaagg tattggaact 5160 tctatttcag atgcgtcaca agctgaggcagaacgacaac aagggatagc tcaggctaaa 5220 tctgatcttc tctccctcca aggagatatcagttcagtca atgatcagat tcaagaactt 5280 cagtatgaac tagttcaatc taaactcgatgagtttgata aaagaattgg agattttgat 5340 gttcggatag caaaagatga gtcaatggctaacagataca cttctgacag caaggaattc 5400 cgaaaataca cctctgatca gaaaaaagctgtggcagagc aagctaaaat ccaacaacaa 5460 aaagttaatt ggattcaaaa agaaattaaaacaaataaag cattgaactc cgctcaacgt 5520 gcacagcttc aagaagagct taaacaggccaagctagatt taatttctgt tcaagaccag 5580 gttcgtgagc tacagaaaca acttgttcaatctaaagttg atgagacact taagtcaatt 5640 gaaaagtcat cttctaaaac ccaagggaaaattaaagatg tcgataacaa aatttcaatg 5700 actgaagaag atgaagacaa ggttaaatactatagcaagc aaataaagct cattcaacaa 5760 caacaaaagg aagcgaagaa atacattaagcagcttgaag aacaaaagaa agctgcgaaa 5820 ggtttccctg acatccagga acagatcactgaagaaatgc aaaactggaa agataaacag 5880 aaagatttta accttgagct ttataacaccaagaagtcga tcaaggatat ctataaatca 5940 ttggctgatg aagttgtatc catctacaaagagatgtacg aaaaaatgcg tgatattgag 6000 ttagaagcgc atcagaaagc gactcaagacttgatcgatg agatagacaa gactgatgac 6060 gaggctaaat ttcaaaaaga attaaaagaaagacaagaca gtattcaaaa gttgactgac 6120 caaattaatc aatactctct tgatgattctgaattcggaa agtcaaaagt caaagaacta 6180 actgaacagc ttcaaaaaga gcagttagaccttgatgatt ttctaaagga tcgcgaaagt 6240 aacaaacgga aagaagcgct ccaagatcagctcgaaaaag atgaggagtc aatcaacaat 6300 aaatacgata atcttgtaaa tgatgaacgagcctttaaaa agcttgagga taagattatg 6360 aatggaaaaa tcaccgatat cgctaagcagcttaatgagt tttctaagtt tattaatacc 6420 aatatggagt ccattggaaa aagtatttcaaacaacctga ttgataaact caaagaagca 6480 tctaatgcac tgaatactgc tgtcaaaggcaacacgacag gtaaaaaagt atcctctttc 6540 gcttctggag ggtacactgg aacaggattaggtgctggta aacttgcatt cctacatgac 6600 aaagaactga tcttaaataa aactgacacagccaacatcc ttgatacggt aaaagctgtt 6660 cgtgaaaccg ctgtggacga ttccccaaaatggggccaag gagtaaaatt agcagacctt 6720 attaaaaaag gaattacttc tattccttcattagttccta acgttaatca atcaatgtta 6780 acaaacagtt taattccaaa tttaaagaagattgagatcc cctcaaaaac aattgcttct 6840 tctggagata aaacaattaa tttaacgaatactttccaca ttgataagct aataggagga 6900 gaatcgggag cgagatcgat gtttgaaagcattaaaaacg aagttgtaaa actaaatggt 6960 agcatgtaag agtctgcaaa agcagactctttatttaact taacttgagg tggaaactca 7020 tgattagaga aagtcaatac tttatgttcaataatatccc ttcttatgaa ttaggagccg 7080 taaatgtaaa tacagaagga 7100 2 2285PRT Bacillius subtilis 2 Leu Ser Gln Asn Leu Lys Ile Ile Leu Thr Pro GlnAla Asp Thr Ser 1 5 10 15 Ser Lys Thr Val Glu Gln Leu Asn Gln Gln IleLys Ser Leu Glu Lys 20 25 30 Lys Leu Asn Ser Leu Lys Leu Asn Thr Asn IleAsp Ser Thr Thr Leu 35 40 45 Lys Ala Leu Gln Glu Phe Ser Ser Ala Ile AspThr Tyr Gln Lys Asn 50 55 60 Leu Lys Ser Tyr Asn Gln Thr Val Lys Glu ThrSer Thr Val Ile Lys 65 70 75 80 Asn Ala Asp Gly Ser Val Glu Lys Leu ThrGln Gln Tyr Lys Lys Asn 85 90 95 Gly Glu Ile Leu Gln Arg Glu Thr Lys IleIle Asn Asn Arg Asn Thr 100 105 110 Ala Leu Lys Gln Glu Thr Gln Glu ValAsn Lys Leu Thr Gln Ala Thr 115 120 125 Glu Lys Leu Gly Gln Val Gln LysLys Thr Val Gln Arg Asn Leu Gln 130 135 140 Gly Gln Pro Thr Lys Val ValGln Lys Asn Arg His Gly Phe Asp Asp 145 150 155 160 Ile Val Tyr Thr ThrAsp Pro Lys Thr Asn Ser Thr Ser Ser Lys Thr 165 170 175 Thr Thr Asn TyrAsp Gln Gln Arg Arg Ala Ile Glu Gln Leu Lys Gln 180 185 190 Asp Leu GluLys Leu Arg Gln Gln Gly Ile Val Thr Asp Thr Thr Ile 195 200 205 Ser SerLeu Gly Arg Lys Ile Asn Thr Ala Gln Ser Ala Gln Gln Ile 210 215 220 GluAla Leu Gln Asn Arg Ile Arg Met Leu Asp Asp Lys Ser Ala Ala 225 230 235240 Val Ala Lys Asn Asn Glu Leu Lys Lys Thr Ile Glu Leu Tyr Gln Arg 245250 255 Gln Ala Gln Val Asn Val Gln Asn Leu Asn Thr Arg Tyr Gly Ser Ser260 265 270 Met Gly Ser Ser Asn Arg Gln Ala Val Gln Asp Tyr Leu Asn AlaVal 275 280 285 Asn Ser Leu Asn Val Ser Thr Gly Ser Asn Asn Ile Arg SerGln Ile 290 295 300 Gln Ser Leu Asn Met Gln Phe Arg Glu Leu Ala Ser AsnAla Gln Thr 305 310 315 320 Ala Ala Asn Gln Ala Ser Ser Phe Gly Ala GluLeu Thr Gln Thr Phe 325 330 335 Lys Ser Met Ser Thr Tyr Leu Ile Ser GlySer Leu Phe Tyr Gly Ala 340 345 350 Ile Ser Gly Leu Lys Glu Met Val SerGln Ala Ile Glu Ile Asp Thr 355 360 365 Leu Met Thr Asn Ile Arg Arg ValMet Asn Glu Pro Asp Tyr Lys Tyr 370 375 380 Asn Glu Leu Leu Gln Glu SerIle Asp Leu Gly Asp Thr Leu Ser Asn 385 390 395 400 Lys Ile Thr Asp IleLeu Gln Met Thr Gly Asp Phe Gly Arg Met Gly 405 410 415 Phe Asp Glu SerGlu Leu Ser Thr Leu Thr Lys Thr Ala Gln Val Leu 420 425 430 Gln Asn ValSer Asp Leu Thr Pro Asp Asp Thr Val Asn Thr Leu Thr 435 440 445 Ala AlaMet Leu Asn Phe Asn Ile Ala Ala Asn Asp Ser Ile Ser Ile 450 455 460 AlaAsp Lys Leu Asn Glu Val Asp Asn Asn Tyr Ala Val Thr Thr Leu 465 470 475480 Asp Leu Ala Asn Ser Ile Arg Lys Ala Gly Ser Thr Ala Ser Thr Phe 485490 495 Gly Val Glu Leu Asn Asp Leu Ile Gly Tyr Thr Thr Ala Ile Ala Ser500 505 510 Thr Thr Arg Glu Ser Gly Asn Ile Val Gly Asn Ser Leu Lys ThrIle 515 520 525 Phe Ala Arg Ile Gly Asn Asn Gln Ser Ser Ile Lys Ala LeuGlu Gln 530 535 540 Ile Gly Ile Ser Val Lys Thr Ala Gly Gly Glu Ala LysSer Ala Ser 545 550 555 560 Asp Leu Ile Ser Glu Val Ala Gly Lys Trp AspThr Leu Ser Asp Ala 565 570 575 Gln Lys Gln Asn Thr Ser Ile Gly Val AlaGly Ile Tyr Gln Leu Ser 580 585 590 Arg Phe Asn Ala Met Met Asn Asn PheSer Ile Ala Gln Asn Ala Ala 595 600 605 Lys Thr Ala Ala Asn Ser Thr GlySer Ala Trp Ser Glu Gln Gln Lys 610 615 620 Tyr Ala Asp Ser Leu Gln AlaArg Val Asn Lys Leu Gln Asn Asn Phe 625 630 635 640 Thr Glu Phe Ala IleAla Ala Ser Asp Ala Phe Ile Ser Asp Gly Leu 645 650 655 Ile Glu Phe ThrGln Ala Ala Gly Ser Leu Leu Asn Ala Ser Thr Gly 660 665 670 Val Ile LysSer Val Gly Phe Leu Pro Pro Leu Leu Ala Ala Val Ser 675 680 685 Thr AlaThr Leu Leu Leu Ser Lys Asn Thr Arg Thr Leu Ala Ser Ser 690 695 700 LeuIle Leu Gly Thr Arg Ala Met Gly Gln Glu Thr Leu Ala Thr Ala 705 710 715720 Gly Leu Glu Ala Gly Met Thr Arg Ala Ala Val Ala Ser Arg Val Leu 725730 735 Lys Thr Ala Leu Arg Gly Leu Leu Val Ser Thr Leu Val Gly Gly Ala740 745 750 Phe Ala Ala Leu Gly Trp Ala Leu Glu Ser Leu Ile Ser Ser PheAla 755 760 765 Glu Ala Lys Lys Ala Lys Asp Asp Phe Glu Gln Ser Gln GlnThr Asn 770 775 780 Val Glu Ala Ile Thr Thr Asn Lys Asp Ser Thr Asp LysLeu Ile Gln 785 790 795 800 Gln Tyr Lys Glu Leu Gln Lys Val Lys Glu SerArg Ser Leu Thr Ser 805 810 815 Asp Glu Glu Gln Glu Tyr Leu Gln Val ThrGln Gln Leu Ala Gln Thr 820 825 830 Phe Pro Ala Leu Val Lys Gly Tyr AspSer Gln Gly Asn Ala Ile Leu 835 840 845 Lys Thr Asn Lys Glu Leu Glu LysAla Ile Glu Asn Thr Lys Glu Tyr 850 855 860 Leu Ala Leu Lys Lys Gln GluThr Arg Asp Ser Ala Lys Lys Thr Phe 865 870 875 880 Glu Asp Ala Ser LysGlu Ile Lys Lys Ser Lys Asp Glu Leu Lys Gln 885 890 895 Tyr Lys Gln IleAla Asp Tyr Asn Asp Lys Gly Arg Pro Lys Trp Asp 900 905 910 Leu Ile AlaAsp Asp Asp Asp Tyr Lys Val Ala Ala Asp Lys Ala Lys 915 920 925 Gln SerMet Leu Lys Ala Gln Ser Asp Ile Glu Ser Gly Asn Ala Lys 930 935 940 ValLys Asp Ser Val Leu Ser Ile Ala Asn Ala Tyr Ser Ser Ile Asp 945 950 955960 Ile Ser Asn Thr Leu Lys Thr Ser Ile Ser Asp Val Val Asn Lys Leu 965970 975 Asn Leu Lys Asp Asp Leu Asp Pro Glu Glu Leu Glu Lys Phe Ser Ser980 985 990 Ser Leu Gly Lys Leu Gln Glu Lys Met Gln Lys Ala Leu Asp SerGly 995 1000 1005 Asp Glu Lys Ala Phe Asp Asn Ala Lys Lys Asp Leu GlnSer Leu Leu 1010 1015 1020 Glu Thr Tyr Ser Lys Ser Asp Ser Ser Ile AspVal Phe Lys Met Ser 1025 1030 1035 104 Phe Asp Lys Ala Gln Lys Asn IleLys Asp Gly Asp Lys Ser Leu Ser 1045 1050 1055 Ser Val Lys Ser Glu ValGly Asp Leu Gly Glu Thr Leu Ala Glu Ala 1060 1065 1070 Gly Asn Glu AlaGlu Asp Phe Gly Lys Lys Leu Lys Glu Ala Leu Asp 1075 1080 1085 Ala AsnSer Val Asp Asp Ile Lys Ala Ala Ile Lys Glu Met Ser Asp 1090 1095 1100Ala Met Gln Phe Asp Ser Val Gln Asp Val Leu Asn Gly Asp Ile Phe 11051110 1115 112 Asn Asn Thr Lys Asp Gln Val Ala Pro Leu Asn Asp Leu LeuGlu Lys 1125 1130 1135 Met Ala Glu Gly Lys Ser Ile Ser Ala Asn Glu AlaAsn Thr Leu Ile 1140 1145 1150 Gln Lys Asp Lys Glu Leu Ala Gln Ala IleSer Ile Glu Asn Gly Val 1155 1160 1165 Val Lys Ile Asn Arg Asp Glu ValIle Lys Gln Arg Lys Val Lys Leu 1170 1175 1180 Asp Ala Tyr Asn Asp MetVal Thr Tyr Ser Asn Lys Leu Met Lys Thr 1185 1190 1195 120 Glu Val AsnAsn Ala Ile Lys Thr Leu Asn Ala Asp Thr Leu Arg Ile 1205 1210 1215 AspSer Leu Lys Lys Leu Arg Lys Glu Arg Lys Leu Asp Met Ser Glu 1220 12251230 Ala Glu Leu Ser Asp Leu Glu Val Lys Ser Ile Asn Asn Val Ala Asp1235 1240 1245 Ala Lys Lys Glu Leu Lys Lys Leu Glu Glu Lys Met Leu GlnPro Gly 1250 1255 1260 Gly Tyr Ser Asn Ser Gln Ile Glu Ala Met Gln SerVal Lys Ser Ala 1265 1270 1275 128 Leu Glu Ser Tyr Ile Ser Ala Ser GluGlu Ala Thr Ser Thr Gln Glu 1285 1290 1295 Met Asn Lys Gln Ala Leu ValGlu Ala Gly Thr Ser Leu Glu Asn Trp 1300 1305 1310 Thr Asp Gln Gln GluLys Ala Asn Glu Glu Thr Lys Thr Ser Met Tyr 1315 1320 1325 Val Val AspLys Tyr Lys Glu Ala Leu Glu Lys Val Asn Ala Glu Ile 1330 1335 1340 AspLys Tyr Asn Lys Gln Val Asn Asp Tyr Pro Lys Tyr Ser Gln Lys 1345 13501355 136 Tyr Arg Asp Ala Ile Lys Lys Glu Ile Lys Ala Leu Gln Gln Lys Lys1365 1370 1375 Lys Leu Met Gln Glu Gln Ala Lys Leu Leu Lys Asp Gln IleLys Ser 1380 1385 1390 Gly Asn Ile Thr Gln Tyr Gly Ile Val Thr Ser ThrThr Ser Ser Gly 1395 1400 1405 Gly Thr Pro Ser Ser Thr Gly Gly Ser TyrSer Gly Lys Tyr Ser Ser 1410 1415 1420 Tyr Ile Asn Ser Ala Ala Ser LysTyr Asn Val Asp Pro Ala Leu Ile 1425 1430 1435 144 Ala Ala Val Ile GlnGln Glu Ser Gly Phe Asn Ala Lys Ala Arg Ser 1445 1450 1455 Gly Val GlyAla Met Gly Leu Met Gln Leu Met Pro Ala Thr Ala Lys 1460 1465 1470 SerLeu Gly Val Asn Asn Ala Tyr Asp Pro Tyr Gln Asn Val Met Gly 1475 14801485 Gly Thr Lys Tyr Leu Ala Gln Gln Leu Glu Lys Phe Gly Gly Asn Val1490 1495 1500 Glu Lys Ala Leu Ala Ala Tyr Asn Ala Gly Pro Gly Asn ValIle Lys 1505 1510 1515 152 Tyr Gly Gly Ile Pro Pro Phe Lys Glu Thr GlnAsn Tyr Val Lys Lys 1525 1530 1535 Ile Met Ala Asn Tyr Ser Lys Ser LeuSer Ser Ala Thr Ser Ser Ile 1540 1545 1550 Ala Ser Tyr Tyr Thr Asn AsnSer Ala Phe Arg Val Ser Ser Lys Tyr 1555 1560 1565 Gly Gln Gln Glu SerGly Leu Arg Ser Ser Pro His Lys Gly Thr Asp 1570 1575 1580 Phe Ala AlaLys Ala Gly Thr Ala Ile Lys Ser Leu Gln Ser Gly Lys 1585 1590 1595 160Val Gln Ile Ala Gly Tyr Ser Lys Thr Ala Gly Asn Trp Val Val Ile 16051610 1615 Lys Gln Asp Asp Gly Thr Val Ala Lys Tyr Met His Met Leu AsnThr 1620 1625 1630 Pro Ser Val Lys Ala Gly Gln Ser Val Lys Ala Gly GlnThr Ile Gly 1635 1640 1645 Lys Val Gly Ser Thr Gly Asn Ser Thr Gly AsnHis Leu His Leu Gln 1650 1655 1660 Ile Glu Gln Asn Gly Lys Thr Ile AspPro Glu Lys Tyr Met Gln Gly 1665 1670 1675 168 Ile Gly Thr Ser Ile SerAsp Ala Ser Gln Ala Glu Ala Glu Arg Gln 1685 1690 1695 Gln Gly Ile AlaGln Ala Lys Ser Asp Leu Leu Ser Leu Gln Gly Asp 1700 1705 1710 Ile SerSer Val Asn Asp Gln Ile Gln Glu Leu Gln Tyr Glu Leu Val 1715 1720 1725Gln Ser Lys Leu Asp Glu Phe Asp Lys Arg Ile Gly Asp Phe Asp Val 17301735 1740 Arg Ile Ala Lys Asp Glu Ser Met Ala Asn Arg Tyr Thr Ser AspSer 1745 1750 1755 176 Lys Glu Phe Arg Lys Tyr Thr Ser Asp Gln Lys LysAla Val Ala Glu 1765 1770 1775 Gln Ala Lys Ile Gln Gln Gln Lys Val AsnTrp Ile Gln Lys Glu Ile 1780 1785 1790 Lys Thr Asn Lys Ala Leu Asn SerAla Gln Arg Ala Gln Leu Gln Glu 1795 1800 1805 Glu Leu Lys Gln Ala LysLeu Asp Leu Ile Ser Val Gln Asp Gln Val 1810 1815 1820 Arg Glu Leu GlnLys Gln Leu Val Gln Ser Lys Val Asp Glu Thr Leu 1825 1830 1835 184 LysSer Ile Glu Lys Ser Ser Ser Lys Thr Gln Gly Lys Ile Lys Asp 1845 18501855 Val Asp Asn Lys Ile Ser Met Thr Glu Glu Asp Glu Asp Lys Val Lys1860 1865 1870 Tyr Tyr Ser Lys Gln Ile Lys Leu Ile Gln Gln Gln Gln LysGlu Ala 1875 1880 1885 Lys Lys Tyr Ile Lys Gln Leu Glu Glu Gln Lys LysAla Ala Lys Gly 1890 1895 1900 Phe Pro Asp Ile Gln Glu Gln Ile Thr GluGlu Met Gln Asn Trp Lys 1905 1910 1915 192 Asp Lys Gln Lys Asp Phe AsnLeu Glu Leu Tyr Asn Thr Lys Lys Ser 1925 1930 1935 Ile Lys Asp Ile TyrLys Ser Leu Ala Asp Glu Val Val Ser Ile Tyr 1940 1945 1950 Lys Glu MetTyr Glu Lys Met Arg Asp Ile Glu Leu Glu Ala His Gln 1955 1960 1965 LysAla Thr Gln Asp Leu Ile Asp Glu Ile Asp Lys Thr Asp Asp Glu 1970 19751980 Ala Lys Phe Gln Lys Glu Leu Lys Glu Arg Gln Asp Ser Ile Gln Lys1985 1990 1995 200 Leu Thr Asp Gln Ile Asn Gln Tyr Ser Leu Asp Asp SerGlu Phe Gly 2005 2010 2015 Lys Ser Lys Val Lys Glu Leu Thr Glu Gln LeuGln Lys Glu Gln Leu 2020 2025 2030 Asp Leu Asp Asp Phe Leu Lys Asp ArgGlu Ser Asn Lys Arg Lys Glu 2035 2040 2045 Ala Leu Gln Asp Gln Leu GluLys Asp Glu Glu Ser Ile Asn Asn Lys 2050 2055 2060 Tyr Asp Asn Leu ValAsn Asp Glu Arg Ala Phe Lys Lys Leu Glu Asp 2065 2070 2075 208 Lys IleMet Asn Gly Lys Ile Thr Asp Ile Ala Lys Gln Leu Asn Glu 2085 2090 2095Phe Ser Lys Phe Ile Asn Thr Asn Met Glu Ser Ile Gly Lys Ser Ile 21002105 2110 Ser Asn Asn Leu Ile Asp Lys Leu Lys Glu Ala Ser Asn Ala LeuAsn 2115 2120 2125 Thr Ala Val Lys Gly Asn Thr Thr Gly Lys Lys Val SerSer Phe Ala 2130 2135 2140 Ser Gly Gly Tyr Thr Gly Thr Gly Leu Gly AlaGly Lys Leu Ala Phe 2145 2150 2155 216 Leu His Asp Lys Glu Leu Ile LeuAsn Lys Thr Asp Thr Ala Asn Ile 2165 2170 2175 Leu Asp Thr Val Lys AlaVal Arg Glu Thr Ala Val Asp Asp Ser Pro 2180 2185 2190 Lys Trp Gly GlnGly Val Lys Leu Ala Asp Leu Ile Lys Lys Gly Ile 2195 2200 2205 Thr SerIle Pro Ser Leu Val Pro Asn Val Asn Gln Ser Met Leu Thr 2210 2215 2220Asn Ser Leu Ile Pro Asn Leu Lys Lys Ile Glu Ile Pro Ser Lys Thr 22252230 2235 224 Ile Ala Ser Ser Gly Asp Lys Thr Ile Asn Leu Thr Asn ThrPhe His 2245 2250 2255 Ile Asp Lys Leu Ile Gly Gly Glu Ser Gly Ala ArgSer Met Phe Glu 2260 2265 2270 Ser Ile Lys Asn Glu Val Val Lys Leu AsnGly Ser Met 2275 2280 2285 3 316 PRT Pseudomonas 3 Pro Lys Val Leu LeuThr Leu Met Val Met Gln Ser Gly Pro Leu Gly 1 5 10 15 Ala Pro Asp GluArg Ala Leu Ala Ala Pro Leu Gly Arg Leu Ser Ala 20 25 30 Lys Arg Gly PheAsp Ala Gln Val Arg Asp Val Leu Gln Gln Leu Ser 35 40 45 Arg Arg Tyr TyrGly Phe Glu Glu Tyr Gln Leu Arg Gln Ala Ala Ala 50 55 60 Arg Lys Ala ValGly Glu Asp Gly Leu Asn Ala Ala Ser Ala Ala Leu 65 70 75 80 Leu Gly LeuLeu Arg Glu Gly Ala Lys Val Ser Ala Val Gln Gly Gly 85 90 95 Asn Pro LeuGly Ala Tyr Ala Gln Thr Phe Gln Arg Leu Phe Gly Thr 100 105 110 Pro AlaAla Glu Leu Leu Gln Pro Ser Asn Arg Val Ala Arg Gln Leu 115 120 125 GlnAla Lys Ala Ala Leu Ala Pro Pro Ser Asn Leu Met Gln Leu Pro 130 135 140Trp Arg Gln Gly Tyr Ser Trp Gln Pro Asn Gly Ala His Ser Asn Thr 145 150155 160 Gly Ser Gly Tyr Pro Tyr Ser Ser Phe Asp Ala Ser Tyr Asp Trp Pro165 170 175 Arg Trp Gly Ser Ala Thr Tyr Ser Val Val Ala Ala His Ala GlyThr 180 185 190 Val Arg Val Leu Ser Arg Cys Gln Val Arg Val Thr His ProSer Gly 195 200 205 Trp Ala Thr Asn Tyr Tyr His Met Asp Gln Ile Gln ValSer Asn Gly 210 215 220 Gln Gln Val Ser Ala Asp Thr Lys Leu Gly Val TyrAla Gly Asn Ile 225 230 235 240 Asn Thr Ala Leu Cys Glu Gly Gly Ser SerThr Gly Pro His Leu His 245 250 255 Phe Ser Leu Leu Tyr Asn Gly Ala PheVal Ser Leu Gln Gly Ala Ser 260 265 270 Phe Gly Pro Tyr Arg Ile Asn ValGly Thr Ser Asn Tyr Asp Asn Asp 275 280 285 Cys Arg Arg Tyr Tyr Phe TyrAsn Gln Ser Ala Gly Thr Thr His Cys 290 295 300 Ala Phe Arg Pro Leu TyrAsn Pro Gly Leu Ala Leu 305 310 315

What is claimed is:
 1. A metalloprotease having the amino acid sequenceshown in SEQ ID NO:
 2. 2. A metalloprotease encoded by the nucleic acidshown in SEQ ID NO:
 1. 3. An expression vector comprising a nucleic acidencoding a gram-positive metalloprotease MP, wherein saidmetalloprotease MP is encoded by the nucleic acid sequence shown in SEQID NO:
 1. 4. A gram-positive microorganism having a mutation or deletionof part or all of the nucleic acid encoding metalloprotease MP, saidmutation or deletion resulting in the inactivation of themetallomrotease MP proteolytic activity, and wherein the metalloproteaseMP has the amino acid sequence shown in SEQ ID NO:
 2. 5. A cleaningcomposition comprising a gram positive microorganism metalloprotease MPwherein the metalloprotease MP has the amino acid sequence shown in SEQID NO:
 2. 6. A cleaning composition comprising a gram positivemicroorganism metalloprotease MP, wherein said metalloprotease MP isencoded by the nucleic acid sequence shown in SEQ ID NO:
 1. 7. Acomposition for the treatment of a textile comprising a metalloproteasecomprising the amino acid sequence as shown in SEQ ID NO:
 2. 8. Acomposition for the treatment of a textile comprising a gram positivemicroorganism metalloprotease MP, wherein said metalloprotease MP isencoded by the nucleic acid sequence shown in SEQ ID NO:
 1. 9. An animalfeed comprising a metalloprotease comprising the amino acid sequenceshown in SEQ ID NO:
 2. 10. An animal feed comprising a gram positivemicroorganism metalloprotease MP, wherein said metalloprotease MP isencoded by the nucleic acid sequence shown in SEQ ID NO: 1.